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Trypanosoma brucei Tim50, a Protein Translocator of Mitochondrial Inner Membrane, Possesses Dual Specific Phosphatase Activity
Author(s) -
Fullerton Marjorie S,
Duncan Melanie R,
Chaudhuri Minu
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.988.12
Subject(s) - phosphatase , trypanosoma brucei , biochemistry , dusp6 , peptide , biology , microbiology and biotechnology , acid phosphatase , alkaline phosphatase , protein tyrosine phosphatase , chemistry , protein phosphatase 2 , enzyme , gene
The single mitochondrion of Trypanosoma brucei is involved in multiple cellular functions. We recently identified a homolog of Tim50, a protein translocator of mitochondrial inner membrane, in T. brucei , which contains a unique CTD‐like phosphatase domain at the C‐terminal. To determine phosphatase activity, we expressed the C‐terminal 6X‐His‐tagged TbTim50 in Escherichia coli and the recombinant protein (rTbTim50) was purified to homogeneity from the bacterial lysate by Ni‐chelation chromatography. The Purified rTbTim50 displayed a phosphatase activity using standard substrate pNPP with a Km of 300 mM. The optimum activity of the TbTim50 phosphatase was observed at pH 5.0. The protein phosphatase activity of rTbTim50 was also measured against phosphotyrosyl and phosphothreonyl peptides. The phosphatase activity of rTbTim50 was two‐fold higher using phosphotyrosyl peptide compared to phosphoserine peptide with Km values 3.75[micro]M and 7.5 [micro]M, respectively. TbTim50 showed similar phosphatase activity to its human Tim50. The effect of site‐directed mutagenesis of two signature CTD phosphatase‐motifs in TbTim50 will be explored. Support by NIH Grant 2Sc1 GM 081146‐05.